Family of canadensol taxanes, the semi-synthetic preparation and therapeutic use thereof

ABSTRACT

The present invention relates to a new taxane having the structure as in formula I and its analogs as depicted in Formula II and Formula III. The invention includes novel semisynthetic and isolation methods for the preparation of these novel taxanes. Specifically, the present invention relates to the new taxane Canadensol isolated from plants of the Taxus genus, in particular,  Taxus canadensis . The present invention further relates to the use of these new taxanes as anticancer agents.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of copending U.S.patent application Ser. No. 08/957,138, filed Oct. 24, 1997, whichclaims priority from Canadian Patent Application 2,188,714, filed Oct.24, 1996, and from Canadian Patent Application 2,197,369, filed Feb. 12,1997. The entire disclosure of each of the aforementioned patentapplications is hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to novel taxanes, semi-synthetic andisolation methods for their production and their use as anticanceragents.

BACKGROUND OF THE INVENTION

[0003] The taxane family of terpenes are considered as an exceptionallypromising group of cancer chemotherapeutic agents. Many taxanederivatives are highly cytotoxic and possess strong in vivo activitiesin a number of leukemic and tumor systems.

[0004] The best known taxane is paclitaxel (Bristol-Myers Squibb's Taxol®). Currently, paclitaxel and semisynthetic analog docetaxel (RhônePoulenc Rorer's Taxotere ®) have been approved for the treatment ofbreast and ovarian cancer, and many clinical trials are underway for anumber of other indications, including lung cancer, Kaposi's sarcoma,and lymphoma.

[0005] A major problem with all of the clinical studies is the limitedavailability of the compounds. Paclitaxel is a natural product which canbe isolated from the bark of Yew trees, but the extraction is difficult,the process is expensive and the yield of paclitaxel is low. Removal ofthe bark destroys Pacific or Western yew, Taxus brevifolia, which islisted among the world's endangered conifer species. The original methodof obtaining Taxol® involved cutting down and stripping tens ofthousands of trees to harvest small amounts of the compound (13,500 kgof Taxus brevifolia bark yields about 1 kg of paclitaxel).

[0006] Alternative modes of obtaining paclitaxel have not solved thesupply problem. The total chemical synthesis of paclitaxel requires morethan thirty steps. Taxus plant cell culture only supplies relatively lowyields of taxanes, and biosynthesis by a yew tree fungus, Taxomycesandreanae has not been sufficient to be economically feasible (NatureBiotech., 14:1055, 1996).

[0007] Isolation of taxanes from the stems and needles of various Taxusspecies offers hope that the supply of taxanes, which can be used forsemi-synthesis, will become more abundant. Because of the structuralcomplexity of paclitaxel, partial synthesis is a far more viableapproach to providing adequate supplies of paclitaxel and paclitaxelprecursors and derivatives than total synthesis. The first successfulpartial synthesis of paclitaxel was developed by Denis et al, (U.S. PatNo. 4,924,011 re-issued as 34,277) using the starting material10-deacetylbaccatin III which can be extracted in relatively high yieldfrom needles of specific species of taxus baccata.

[0008] Many derivatives of paclitaxel have also been synthesized sincethe realization of its utility as a therapeutic agent in the treatmentof cancer. Examples include 7-deoxy-taxol analogs (as described in PCTApplication No. WO 94/13655), which are useful for breast, ovarian,lung, colonic and gastric cancers, malignant melanoma and leukemia. Anumber of benzoate derivatives (Canadian Patent Application No.2,095,375), have also been synthesized. Paclitaxel analogs with a ketonemoiety (U.S. Pat. No. 5,440,047 and PCT Application No. WO 95/33736),have also been reported to have remarkable antitumoral and antileukemicproperties. Derivatives with an epoxide residue have also been disclosed(PCT Application No.WO /94/13654) which exhibit antitumour activity. Thepreparation of a further group of taxane derivatives have been describedin PCT Application Nos. WO 95/02400 and WO 93/21173 which arestructurally very similar to paclitaxel but have somewhat increasedchemotherapeutic activity when compared to paclitaxel.

[0009] Although the use of paclitaxel is successful against a number ofspecific tumor types, it is not universally effective. Different taxanesexhibit different efficacies to various tumor types, hence there is anurgent need for novel compounds from the taxane family that are closelyrelated to paclitaxel in their chemical structures but which have morepotent chemotherapeutic activities.

SUMMARY OF THE INVENTION

[0010] One embodiment of the present invention is to provide a newtaxane and its derivatives, which have chemotherapeutic activities.

[0011] A further embodiment of the present invention is to provide acompound, hereinafter referred to as Canadensol, having the followingstructural Formula I:

[0012] a previously unknown constituent of the Canada yew, Taxuscanadensis.

[0013] It is another embodiment of the invention to provide analogshaving in common with Canadensol the following structural Formula II:

[0014] as well as a process for preparing such compounds from thestarting material baccatin III.

[0015] Wherein: R₁ in Formula II is selected from the group consistingof:

[0016] C₁-C₆ alkyl including methyl, isopropyl, isopropenyl, propenyl,butyl, cyclopropyl, substituted alkyl including halo,di-(tri-halomethyl)methyl, 3′-trihalo-n-propyl, or phenyl substitutedwith: one, 2 or 3 C₁-C₄ alkyl, C₁-C₃ alkoxy, halo, C₁-C₃ alkylthio,trifluoromethyl, C₂-C₆ dialkylamino, hydroxy or nitro, 2-furyl,2-thienyl; 1-napthyl, 2-napthyl, isopropoxy, isopropenoxy, propenoxy,cyclopropoxy, di-(trihalomethyl)methoxy, 3′-trihalo-n-propanoxy,dimethylamino, ethylamino, isopropylamino, propylamino, isopropenamino,imidazol, acetyl, hydroxycarbonyl, 2-(hydroxy)ethyl, 2-(methyl)-propyl,benzyl.

[0017] Wherein: R₂ in Formula II is selected from the group consistingof: —OAc or —OH.

[0018] It is a further embodiment of the present invention to provideanalogs having in common with Canadensol the following structuralFormula III:

[0019] as well as a process for preparing such compounds from thestarting material baccatin III.

[0020] Wherein: R₃ in Formula III is selected from the group consistingof:

[0021] C₁-C₆ alkyl including methyl, isopropanoyl, isopropenoyl,propenoyl, cyclopropanoyl, substituted alkyl including halo,di-(tri-halomethyl)acetanoyl, 3′-trihalo-n-propanoyl,3,4-methylene-dioxyphenyl or phenyl substituted with one, 2 or 3 C₁-C₄alkyl, C₁-C₃ alkoxy, halo, C₁-C₃ alkylthio, trifluoromethyl, C₂-C₆dialkylamino, hydroxy or nitro, 2-furyl, 2-thienyl, 1-napthyl,2-napthyl, isopropoxycarbonyl, isopropenoxycarbonyl, propenoxycarbonyl,cyclopropoxycarbonyl, di-(trihalomethyl)methoxycarbonyl,3′-trihalo-n-propanoxycarbonyl, dimethylaminocarbonyl,ethylaminocarbonyl, isopropylaminocarbonyl, propylaminocarbonyl,isopropenaminocarbonyl, imidazolcarbonyl, pyruvyl, oxalyl,2-(hydroxy)ethylcarbonyl, 2-(methyl)-propylcarbonyl, benzylcarbonyl.

[0022] Due to the immediate requirement for additional novel taxanes, afurther object of the present invention is to provide a family oftaxanes, represented by Formula II and Formula III. These compounds areuseful for the treatment of, or in the preparation of paclitaxelderivatives for use in treatment of, cancers and leukemias. Theinvention also provides for pharmaceutically acceptable compositionscompounds of Formula II and Formula III, for use as therapeutic agentsfor use as anticancer agents in the management of such disease.

[0023] It is a further embodiment of this invention to provide a simpleand inexpensive semi-synthetic method for the production of these noveltaxanes. Accordingly, it is an object of this invention to provide areproducible method for the isolation and semi-synthesis of taxanes fromplant matter derived from the Taxus genus of plants.

[0024] Yet a further object of the present invention is to provide, inparticular a method for the isolation and semi-synthesis of a noveltaxane, named Canadensol, which exhibits higher chemotherapeuticactivity than paclitaxel.

[0025] It is a further object of this invention to provide a method forthe production of a number of protected intermediates, that are usefulin the semi-synthesis of novel taxanes.

[0026] It is yet a further embodiment of the present invention toprovide for the use of the compounds exhibited in Formula I, II, and IIIin the treatment of various maladies, including various forms of cancer.

[0027] These and other objectives, as well as the nature, scope andutilization of this invention, will become readily apparent to thoseskilled in the art from following the description, the drawings and theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention is disclosed in connection with theappended drawings, in which:

[0029]FIG. 1 shows the results of a microtubule assay indicating thatCanadensol exhibits a higher antimitotic activity than paclitaxel at thesame concentration;

[0030]FIG. 2 shows the results of a microtubule assay demonstrating aconcentration dependent antimitotic effect of Canadensol and thatCanadensol is more active than paclitaxel at the same concentration;

[0031]FIG. 3 demonstrates in a microtubule assay that, at the sameconcentration, paclitaxel and taxcultine exhibit similar antimitoticactivity, but in the same experiment, Canadensol is more effective;

[0032]FIG. 4 shows the activity of semi-synthetic Canadensol versus thatof a natural product prior to removing the taxcultine in a microtubuleassay;

[0033]FIG. 5 shows the NMR data of N-debenzoyl-N-n-pentanoyl-paclitaxel;

[0034]FIG. 6 shows the in vitro cytotoxic effects of taxoids(Paclitaxel, Canadensol, and Taxcultine) on A2780 cells versus taxoidconcentration;

[0035]FIG. 7 shows the in vitro cytotoxic effects of Paclitaxel onA2780, MCF7 and DA3 cells verses Paclitaxel concentration;

[0036]FIG. 8A shows the perturbation of tumour volume with differingdoses of taxol™ versus time,

[0037]FIG. 8B shows the perturbation of body weight with differing dosesof taxol™ versus time; and

[0038]FIG. 9 shows the perturbation of tumour volume with differingdoses of Canadensol and a control sample versus time.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention comprises the compound Canadensol as wellas derivatives thereof, having the structure Formula II and Formula III:

[0040] Wherein: R₁ in Formula II is selected from the group consistingof:

[0041] C₁-C₆ alkyl including methyl, isopropyl, isopropenyl, propenyl,propyl, butyl, cyclopropyl, substituted alkyl including halo,di-(tri-halomethyl)methyl, 3′-trihalo-n-propyl, or phenyl substitutedwith: one, 2 or 3 C₁-C₄ alkyl, C₁-C₃ alkoxy, halo, C₁-C₃ alkylthio,trifluoromethyl, C₂-C₆ dialkylamino, hydroxy or nitro, 2-furyl,2-thienyl; 1-napthyl, 2-napthyl, isopropoxy, isopropenoxy, propenoxy,cyclopropoxy, di-(trihalomethyl)methoxy, 3′-trihalo-n-propanoxy,dimethylamino, ethylamino, isopropylamino, propylamino, isopropenamino,imidazol, acetyl, hydroxycarbonyl, 2-(hydroxy)ethyl, 2-(methyl)-propyl,benzyl.

[0042] Wherein: R₂ in Formula III is selected from the group consistingof: —OAc or —OH.

[0043] It is also another object of the invention to provide analogshaving in common with Canadensol the following structural Formula III:

[0044] as well as a process for preparing such compounds from thestarting material baccatin III.

[0045] Wherein: R₃ in Formula III is selected from the group consistingof:

[0046] C₁-C₆ alkyl including methyl, propyl, isopropyl, butyl,1′-methyl-propyl, 2′-methyl-propyl, pentyl and all possiblestereoisomers; isopropanoyl; isopropenoyl, propenoyl, cyclopropanoyl,substituted alkyl including halo, di-(tri-halomethyl)acetanoyl,3′-trihalo-n-propanoyl, 3,4-methylene-dioxyphenyl or phenyl substitutedwith one, 2 or 3 C₁-C₄ alkyl, C₁-C₃ alkoxy, halo, C₁-C₃ alkylthio,trifluoromethyl, C₂-C₆ dialkylamino, hydroxy or nitro, 2-furyl,2-thienyl, 1-napthyl, 2-napthyl, isopropoxycarbonyl,isopropenoxycarbonyl, propenoxycarbonyl, cyclopropoxycarbonyl,di-(trihalomethyl)methoxycarbonyl, 3′-trihalo-n-propanoxycarbonyl,dimethylaminocarbonyl, ethylaminocarbonyl, isopropylaminocarbonyl,propylaminocarbonyl, isopropenaminocarbonyl, imidazolcarbonyl, pyruvyl,oxalyl, 2-(hydroxy)ethylcarbonyl, 2-(methyl)-propylcarbonyl,benzylcarbonyl.

[0047] The following definitions apply to these compounds and throughoutthe present disclosure.

[0048] The term “alkyl” as used herein refers to a monovalent groupderived by the removal of a single hydrogen atom from a straight- orbranched-chain saturated hydrocarbon containing one to six atomsincluding, but not limited to, methyl, ethyl, n- and iso-propyl, n-,sec-, iso- and tert-butyl, pentyl and hexyl.

[0049] The term “substituted alkyl” as used herein refers to an alkylgroup as defined above substituted with between one and three groupssuch as hydroxyl, sulfhydryl, alkoxyl, carboxyl and halogen.

[0050] The term “alkoxy” as used herein refers to an alkyl function asdefined above attached via an oxygen atom including, but not limited to,methoxy, ethoxy, iso-propoxy, butoxy and tert-butoxy.

[0051] The term “substituted alkoxy” as used herein refers to an alkoxygroup as defined above substituted with between one and three groupssuch as hydroxyl, sulfhydryl, alkoxyl, thioalkoxyl, carboxyl, amino andhalogen.

[0052] The term “alkoxyalkyl” as used herein refers to an alkoxyfunction as defined above attached to an alkyl group including, but notlimited to, methylpropionoyl and ethylbutanoyl.

[0053] The term “alkanoyl” as used herein refers to an alkyl function asdefined above attached via a carbonyl group including, but not limitedto, acetoyl, propionyl, butanoyl and isobutanoyl.

[0054] The term “hydroxyalkyl” as used herein refers to an alkylfunction as defined above substituted with between one to three hydroxylgroups including, but not limited to, hydroxyethyl and hydroxypropyl.

[0055] The term “cycloalkyl” as used herein refers to an alkyl functionas defined above where some of the carbon atoms are joined to form aring including, but not limited to, cyclopropyl, cyclobutyl andcyclopentyl.

[0056] The term “alkenyl” as used herein refers to an alkyl function asdefined above which contains between one and three unsaturated bondsincluding, but not limited to, isopropenyl.

[0057] The term “phenoxy” as used herein refers to an phenyl group asdefined above attached via an oxygen atom.

[0058] The term “aminoalkyl” as used herein refers to an alkyl functionas defined above substituted with amino or substituted amino, as definedelsewhere herein.

[0059] The term “aminoalkanoyl” as used herein refers to an alkanoylfunction as defined above substituted with between one and three aminogroups including, but not limited to, 2-aminopropanoyl, 4-aminobutanoyl,and 6-aminohexanoyl. Additionally, the amino groups may optionally besubstituted with peptidyl residues formed therefrom.

[0060] The term “substituted alkoxyalkyl” as used herein refers to analkoxyalkyl group as defined above substituted with between one andthree groups such as hydroxyl, sulfhydryl, alkoxyl, thioalkoxyl,carboxyl, amino and halogen.

[0061] The term “substituted alkanoyl” as used herein refers to analkanoyl group as defined above substituted with between one and threegroups such as hydroxyl, sulfhydryl, alkoxyl, carboxyl and halogen.

[0062] The term “substituted hydroxyalkyl” as used herein refers to anhydroxyalkyl group as defined above substituted with between one andthree groups such as sulphydryl, carbonyl and halogen.

[0063] The term “substituted cycloalkyl” as used herein refers to ancycloalkyl group as defined above substituted with between one and threegroups such as hydroxyl, sulfhydryl, alkoxyl, thioalkoxyl, carboxyl,amino and halogen.

[0064] The term “substituted alkenyl” as used herein refers to analkenyl group as defined above substituted with between one and threegroups such as hydroxyl, sulfhydryl, alkoxyl, thioalkoxyl, carboxyl,amino and halogen.

[0065] The term “substituted phenyl” as used herein refers to a phenylgroup substituted with between one and three substituents independentlyselected from alkyl, halogen, haloalkyl, alkoxy, benzyloxy, thioalkoxy,hydroxy, alkanoyl, carboxy, amino, alkylamino, dialkylamino, nitro and—OSO₃H.

[0066] The term “substituted phenoxy” as used herein refers to a phenoxygroup substituted with between one and three substituents independentlyselected from alkyl, halogen, haloalkyl, alkoxy, benzyloxy, thioalkoxy,hydroxy, alkanoyl, carboxy, amino, alkylamino, dialkylamino, nitro and—OSO₃H.

[0067] The term “substituted amino” as used herein refers to an aminogroup substituted with one or two alkyl groups including, but notlimited to, t-butylamino, benzylamino and N,N-dimethylamino.

[0068] The term “substituted aminoalkyl” as used herein refers to anaminoalkyl group substituted with between one and three groupsincluding, but not limited to, hydroxyl, sulfhydryl, alkoxyl,thioalkoxyl, carboxyl, amino and halogen.

[0069] The term “substituted aminoalkanoyl” as used herein refers to anaminoalkanoyl group substituted with between one and three groupsincluding, but not limited to, hydroxyl, sulfhydryl, alkoxyl,thioalkoxyl, carboxyl, amino and halogen.

[0070] The term “substituted imidazol” as used herein refers to animidazol group substituted with between one and three groups including,but not limited to, hydroxyl, sulfhydryl, alkoxyl, thioalkoxyl,carboxyl, amino and halogen.

[0071] The term “halogen” as used herein refers to a group selected frombromo (Br), chloro (Cl), fluoro (F) and iodo (I).

[0072] The term “haloalkyl” as used herein refers to an alkyl group asdefined above substituted with between one and three halogen atomsincluding, but not limited to, fluoromethyl, trifluoromethyl andfluoroethyl.

[0073] The term “acylated” as used herein refers to an alkyl groupattached to a carbonyl.

[0074] The term “protecting group” as used herein is a term well-knownin the art and refers to substituents on functional groups of compoundsundergoing chemical transformation which prevent undesirable reactionsand degradations during a synthesis; see, for example, T. H. Greene,“Protective Groups in Organic Synthesis”, John Wiley and Sons (1981).

[0075] The term “N-protected” and “N-protecting” as used herein refer tothe use of a group intended to protect an amino function or theN-terminus of an amino acid or peptide against undesirable reactionsduring a synthetic procedure or to prevent the attack of exopeptidaseson the compound or to increase the solubility of the compound andincludes, but is not limited to, such uses sulfonyl; acyl, such asacetyl, pivaloyl and benzoyl; alkoxycarbonyl, such astert-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz); and L- orD-aminoacyl residues, which may themselves be N-protected. Otherexamples may be found in ‘The Peptides’ E. Gross and J. Meinenhofer, Vol3, Academic Press (1981).

[0076] It will be appreciated by those skilled in the art that thecompounds of Formula II and Formula III, depending on the substituents,may contain one or more chiral centers and thus exist in the form ofmany different isomers, optical isomers (i.e. enantiomers) and mixturesthereof including racemic mixtures. All such isomers, enantiomers andmixtures thereof including racemic mixtures are included within thescope of the invention.

[0077] The compound of Formula I, Canadensol, has been isolated from theneedles of the Taxus genus. The vegetal material preferably consists ofthe needles which is rapidly regenerated and therefore in abundantsupply, though other material such as the roots or the bark of the Taxusbushes may be used.

[0078] Early attempts at the recovery of Canadensol from thechromatographic fractions containing it, consisted of numerous reversephase HPLC steps resulting in low yields.

[0079] The compound of Formula I is extracted by means of chlorinatedsolvents, for example dichloromethane, in admixture with alcohols suchas methyl alcohol. The purification of Canadensol requires apurification through column chromatography, wherein silica gel ispreferably used as the stationary phase. Solvent mixtures, consisting ofan aliphatic hydrocarbon, for example, hexane with a chlorinatedsolvent, such as dichloromethane, together with a higher polaritysolvent, such as ethyl acetate or acetone are used as eluents.

[0080] The preferred compounds of the present invention can also besynthesized using a new and unique combination of conventionalpreparative steps and recovery methods known to those skilled in the artof organic and bio-organic synthesis, while providing a new and uniquecombinations for the overall synthesis of each compound. Preferredsynthetic routes for intermediates involved in the synthesis as well asthe resulting taxane derivative compounds of the present inventionfollow. Successful preparation of these compounds is possible by way ofseveral synthetic routes: two of which are outlined in Schemes I and II(demonstrated in Examples I and II, correspondingly).

[0081] Briefly, the semi-synthesis methods use baccatin III as startingmaterial, which can be obtained from a number of its derivatives, one ofthe preferred derivatives is 10-deacetylbaccatin III. The conversion of10-deacetylbaccatin III into baccatin III can be achieved according tothe technique described in Denis J N, et al., J. Am. Chem. Soc.,110:5917 (1988). The conversion of baccatin III into taxol derivativesrequires that the hydroxyl moiety at C-7 be protected prior toderivatization with the appropriate side chain at C-13. The appropriateside chains can be synthesized from alkyl cinnamate. See Denis J N, etal., J. Org. Chem. 55:1959 (1990) for an example of a suitable sidechain synthesised from methyl cinnamate.

[0082] Scheme I

[0083] Step A

[0084] Compound (i); baccatin III, is derivatized at C-7 by reactingwith Compound (ii); a suitable protecting group (X). It is necessary toprotect the hydroxyl group at position 7 of baccatin III, to preventcoupling at C-7 and/or racemization at C-7 (through retro-aldolreaction). This can be achieved through the use of silyl chlorides (forexample from triethyl, tri-isopropyl, t-butyldimethyl ort-butyldiphenyl) or alkyl chlorides (for example from benzyl chloride,methoxy-methyl chloride, allyl chloride or methoxy-ethyl chloride) or bythe use of dihydropyran. The above reaction yields Compound (iii); a7-protected baccatin III intermediate.

[0085] Step B

[0086] The 7-protected baccatin III intermediate, Compound (iii), isthen coupled at C-13 by the use of an appropriate acid, Compound (iv).The appropriate acid can be synthesized from alkyl cinnamate. The iiabove coupling procedure generates a taxol derivative with a protectinggroup at C-7 as illustrated in Compound (v).

[0087] Step C

[0088] This step achieves two functions: the protecting group at C-7 isremoved, and the cyclized side chain at C-13 is opened-up and the sidechain amine is acylated. This transformation, into Compound (vi), can beachieved through the use of an organic acid, followed by acylation usingbicarbonate salts and an acyl chloride. In Compound (vi), R₁ is selectedfrom the group consisting: C₁-C₆ alkyl including methyl, isopropyl,isopropenyl, propenyl, butyl, cyclopropyl, substituted alkyl includinghalo, di-(trihalomethyl)methyl, 3′-trihalo-n-propyl, or phenylsubstituted with: one, 2 or 3 C₁-C₄ alkyl, C₁-C₃ alkoxy, halo, C₁-C₃alkylthio, trifluoromethyl, C₂-C₆ dialkylamino, hydroxy or nitro,2-furyl, 2-thienyl; 1-napthyl, 2-napthyl, isopropoxy, isopropenoxy,propenoxy, cyclopropoxy, di-(trihalomethyl)methoxy,3′-trihalo-n-propanoxy, dimethylamino, ethylamino, isopropylamino,propylamino, isopropenamino, imidazol, acetyl, hydroxycarbonyl,2-(hydroxy)ethyl, 2-(methyl)propyl, benzyl.

[0089] Where R₁ is isopropyl, this scheme can yield 0.86% (See Example1).

[0090] Scheme II

[0091] Step A

[0092] Compound (i); baccatin III, is derivatized at C-7 by reactingwith Compound (vii) a suitable protecting group (Y). It is necessary toprotect the hydroxyl group at position 7 of baccatin III, to preventcoupling at C-7 and/or racemization at C-7 (through a retro-aldolreaction). This can be achieved through the use of chloroalkyl formates(for example from trichloroethyl chloroformate,trichloromethylchloroformate, dichloroethyl-chloroformate,dichloromethyl chloroformate, etc.). The above reaction yields Compound(viii); a 7-protected baccatin III intermediate. When Y is —CO₂CH₂CCl₃,the yield of Compound (viii) is 25.5%.

[0093] Step B

[0094] The 7-protected baccatin III intermediate, Compound (viii), isthen coupled at C-13 by the use of the acid, Compound (iv) which can besynthesized from alkyl cinnamate. The above coupling procedure generatesa taxol derivative with a protecting group at C-7 as illustrated inCompound (ix). When Y is —CO₂CH₂CCl₃, the coupling reaction withcompound (iv) affords Compound (ix) in 53.3% yield.

[0095] Step C

[0096] In Compound (x), R₁ is selected from the group consisting: C₁-C₆alkyl including methyl, isopropyl, isopropenyl, propenyl, butyl,cyclopropyl, substituted alkyl including halo,di-(tri-halomethyl)methyl, 3′-trihalo-n-propyl, or phenyl substitutedwith: one, 2 or 3 C₁-C₄ alkyl, C₁-C₃ alkoxy, halo, C₁-C₃ alkylthio,trifluoromethyl, C₂-C₆ dialkylamino, hydroxy or nitro, 2-furyl,2-thienyl; 1-napthyl, 2-napthyl, isopropoxy, isopropenoxy, propenoxy,cyclopropoxy, di-(trihalomethyl)methoxy, 3′-trihalo-n-propanoxy,dimethylamino, ethylamino, isopropylamino, propylamino, isopropenamino,imidazol, acetyl, hydroxycarbonyl, 2-(hydroxy)ethyl, 2-(methyl)-propyl,benzyl.

[0097] In this step the cyclized side chain at C-13 is opened-up. Thistransformation, into Compound (x), can be achieved through the use of anorganic acid, followed by alkylation using bicarbonate salts and an acylchloride.

[0098] Step D

[0099] The protecting group at C-7 is then removed from Compound (x) bythe use of reducing agents such as zinc in the presence of acetic acidand methanol to yield Compound (vi). When R₁ is isopropyl, this schemecan yield 74.7% (See Example 2).

[0100] One particular advantage of this invention is that the startingmaterial, baccatin III, can be obtained from 10-deacetylbaccatin IIIwhich is produced in relatively high concentrations in the needles ofTaxus canadensis. A further important consideration when choosingstarting materials is that they must be available in relatively largequantities and be a rapidly renewable resource. 10-deacetyl baccatin IIIsatisfy this criteria as they can be isolated by simplerecrystallisations and preparative reverse phase HPLC instead of manysilica gel columns, hence the yields are relatively high.

[0101] The starting material for use in this invention is vegetalmaterial, selected from a group of plants commonly referred to astaxads. The most suitable plants of this group are the species Taxus.The semi-synthetic method disclosed is effective when using the roots,bark or leaves of the Taxus plant but we consider it prudent to use asource that is rapidly regenerated (such as the leaves i.e. needles) andtherefore in abundant supply. Amongst the Taxus species, Taxuscanadensis is a preferred source for use in the semi-synthesis of thenovel taxanes claimed in this invention. Thus one useful aspect of thecurrent invention is largely dependent upon an abundant supply ofbaccatin III which can be derived from 10-deacetylbaccatin III.

[0102] Paclitaxel derivatives are useful for their antitumor activity,particularly for the treatment of the same cancers for which taxol hasbeen shown active, including human lung tumors, melanoma, leukemia,mammary tumors, and colon cancer. We anticipate that the taxolderivatives of the current invention will have utility in the treatmentof the same cancers for which taxol has been shown to be active.

[0103] Microtubules are an integral part of eukaryotic cells, andmicrotubule assembly is importantly associated with cell division andmultiplication. Known antitumor compounds have been studied for theireffect on microtubule assembly. For example, vinca alkaloids, such asvinblastine and vincristine have been shown to disrupt cellularmicrotubules, i.e. in vitro they have been shown to inhibit microtubuleassembly and to depolymerize steady state microtubules. Similarly,colchicine has been shown to depolymerize microtubules in cells.

[0104] Paclitaxel has been shown to exhibit a very unique mechanism ofaction, in that it promotes the assembly of microtubules, but inhibitstheir disassembly, thereby interfering with the G2 and M phases of cellcycles and division. In vitro studies have shown that microtubules, oncepolymerized, in the presence of taxol resists depolymerization by otheragents such as CaCl₂ or cold temperature which normally depolymerizemicrotubules.

[0105] The present inventors have conducted studies to investigate theeffect of derivatives of taxol on the microtubule assembly. Themicrotubule assembly study was conducted using Canadensol according tothe in vitro procedures disclosed, for example in Parness et al., J.CellBiol. 91:479 1981. In these studies, conditions can be establishedwhereby a dynamic steady state exists between tubulin assembling intomicrotubules and the disassembly at the other end. This dynamic steadystate can be measured spectrophotometrically, observing the absorbanceof the solution at 350 nm. As stated above, it has been shown that taxolbinds specifically and reversibly to the protein, stabilizing themicrotubules in the polymerized form. This effect can be visualized byfollowing the increase in absorbance of the solution at 350 nm. Cellstreated with taxol, or derivatives thereof which exhibitchemotherapeutic activity, usually die as they are effectively blockedin mitosis.

[0106] The results presented in FIGS. 1-4, indicate that compounds ofFormula II (eg. when R₁ is isopropyl or propyl) exhibit excellenttubulin stabilizing activity. These compounds, therefore, are usefulantitumor agents due to their biological activity. It is therefore oneembodiment of the present invention to claim the use of the twocompounds of Formula II, when R₁ is isopropyl and propyl, as anticanceragents.

[0107] The present invention also provides pharmaceutical compositionscontaining a compounds as disclosed in the claims in combination withone or more pharmaceutically acceptable, inert or physiologicallyactive, diluents or adjuvants. The compounds of the invention can befreeze dried and, if desired, combined with other pharmaceuticallyacceptable excipients to prepare formulations for administration. Thesecompositions may be presented in any form appropriate for theadministration route envisaged. The parenteral and the intravenous routeare the preferential routes for administration.

[0108] Compounds of the general Formula II may be administered orally,topically, parenterally, by inhalation or spray or rectally in dosageunit formulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. The term parenteral as usedherein includes subcutaneous injections, intravenous, intramuscular,intrasternal injection or infusion techniques. In addition, there isprovided a pharmaceutical formulation comprising a compound of generalFormula II and a pharmaceutically acceptable carrier. One or morecompounds of general Formula II may be present in association with oneor more non-toxic pharmaceutically acceptable carriers and/or diluentsand/or adjuvants and if desired other active ingredients. Thepharmaceutical compositions containing compounds of general Formula IImay be in a form suitable for oral use, for example, as tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsion hard or soft capsules, or syrups or elixirs.

[0109] Compositions intended for oral use may be prepared according toany known to the art for the manufacture of pharmaceutical compositionsand such compositions may contain one or more agents selected from thegroup consisting of sweetening agents, flavouring agents, colouringagents and preserving agents in order to provide pharmaceuticallyelegant and palatable preparations. Tablets contain the activeingredient in admixture with non-toxic pharmaceutically acceptableexcipients which are suitable for the manufacture of tablets. Theseexcipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate: granulating and disintegrating agents for example, cornstarch, or alginic acid: binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate may be employed.

[0110] Formulations for oral use may also be presented as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredient is mixed withwater or an oil medium, for example peanut oil, liquid paraffin or oliveoil.

[0111] Aqueous suspensions contain active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia:dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethyene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample hepta-decaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxy-benzoate, one or more coloringagents, one or more flavoring agents or one or more sweetening agents,such as sucrose or saccharin.

[0112] Oily suspensions may be formulated by suspending the activeingredients in a vegetable oil, for example arachis oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.The oily suspensions may contain a thickening agent, for examplebeeswax, hard paraffin or cetyl alcohol. Sweetening agents such as thoseset forth above, and flavoring agents may be added to provide palatableoral preparations. These compositions may be preserved by the additionof an anti-oxidant such as ascorbic acid.

[0113] Dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water provide the activeingredient in admixture with a dispersing or wetting agent, suspendingagent and one or more preservatives. Suitable dispersing or wettingagents and suspending agents are exemplified by those already mentionedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present.

[0114] Pharmaceutical compositions of the invention may also be in theform of oil-in-water emulsions. The oils phase may be a vegetable oil,for example olive oil or arachis oil, or a mineral oil, for exampleliquid paraffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monoleate, and condensation products ofthe said partial esters with ethylene oxide, for example polyoxyethylenesorbitan monoleate. The emulsions may also contain sweetening andflavoring agents.

[0115] Syrups and elixirs may be formulated with sweetening agents, forexample glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions may be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension may be formulation according to known art using thosesuitable dispersing or wetting agents and suspending agents which havebeen mentioned above. The sterile injectable preparation may also besterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

[0116] The compound(s) of the general Formula I, Formula II and FormulaIII may be administered, together or separately, in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are cocoa butter and polyethylene glycols.

[0117] Compound(s) of general Formula I, Formula II, and Formula III maybe administered, together or separately, parenterally in sterile medium.The drug, depending on the vehicle and concentration used, can either besuspended or dissolved in the vehicle. Advantageously, adjuvants such aslocal anaesthetics, preservatives and buffering agents can be dissolvedin the vehicle.

[0118] The mode, dosage and schedule of administration of taxol in humancancer patients has been studied extensively (see Ann. Int. Med. 111:2731989). For the compounds of this invention, the dose to be administered,whether a single dose, multiple dose, or a daily dose, will vary withthe particular compound being used. Factors to consider when decidingupon a dose regimen include potency of the compound, route ofadministration, size of the recipient and the nature of the patient'scondition.

[0119] The dosage to be administered is not subject to defined limits,but in will usually be an effective amount. It will usually be theequivalent, on a molar basis of the pharmacologically active free formproduced from a dosage formulation upon the metabolic release of theactive free drug to achieve its desired pharmacological andphysiological effects.

[0120] An oncologist skilled in the art of cancer treatment will be ableto ascertain, without undue experimentations, appropriate protocols foreffective administration of the compounds of this present invention byreferring to the earlier studies of taxol and its derivatives.

[0121] Particular reagents and conditions utilized in these synthesesare described in detail in the Examples which follow. Also, it will beappreciated by one skilled in the art that selective protection anddeprotection steps affecting the several hydroxyl groups on the baccatinIII structure may be carried out in varying order or number of steps, asnecessary, and that Schemes I and II are intended to encompass suchvariations.

[0122] The Taxus canadensis plant was extracted as reported previously(L. O. Zamir, M. E. Nedea, Z.-H. Zhou, S. Bélair, G. Caron, F. Sauriol,E. Jacqmain, F. I. Jean, F.-X. Garneau and O. Mamer, Can. J. Chem.,73:655, 1995), the additional chromatographic purification step leadingto a new taxane with a side chain, never isolated before in Taxus spp.is described below.

[0123] Isolation of Taxanes with Reverse Phase HPLC: Taxanes in thebrown solid are separated on a preparative HPLC using an ODS-2 reversephase column (2.0×50 cm; Whatman) and a Waters Delta Prep 3000instrument coupled to a model 481 variable wavelength detector at 227nm. The products are eluted with a gradient over 140 minutes ofacetonitrile:water (25:75) to 100% acetonitrile. At 55.5 mm, a peakcomprising 10 hydroxyacetylbaccatin VI, among other taxanes iscollected.

[0124] Isolation of N-debenzoyl-N-n-pentanoylpaclitaxel with ReversePhase HPLC: The peak at 55.5 minutes was found to contain the compoundN-debenzoyl-N-n-pentanoylpaclitaxel. In order to isolate this compound,the 55.5 minute peak was separated further utilizing the samepreparative HPLC system as described above. However, this separationused a gradient over 50 minutes of isopropanol:water (25:75) to 100%isopropanol at a flow rate of 6 mL per minute. The largest peak fromthis gradient was centered at a retention time of 41 minutes. Theshoulder of this 41 minute-centered peak, from 44 to 48 minutes, wascollected and purified further using the same HPLC system eluting with agradient over 100 minutes of isopropanol:water (25:75) to 100%isopropanol at a flow rate of 7 mL per minute. The product eluting at 55minutes was collected. Analysis of the product on analytical HPLCyielded a single pure product eluting at 32.9 minutes when using agradient over 50 minutes of acetonitrile:water (25:75) to 100%acetonitrile. The structure of N-debenzoyl-N-n-pentanoylpaclitaxel waselucidated by NMR analysis, the results being listed in FIG. 5.

[0125] Further, to assist in understanding the current invention, thefollowing non-limiting examples are provided. The following examplesshould not be construed as specifically limiting the present invention,variations presently known or later developed, which would be in theunderstanding of one skilled in the art and considered to fall withinthe scope of the present invention as described herein.

EXAMPLE 1 Semi-Synthesis of Canadensol (Scheme I)

[0126] The preparation of Compound 1 was carried out through eight stepreactions from methyl cinnamate (as described in Denis J. D., Correa A.,Greene A. E., J. Org. Chem., 55:1959, 1990). The preparation of Compound2 was carried out through two step reactions from 10-deacetyl baccatinIII (as described in Denis J. N., Greene A. E., Guenard D.,Gueritte-Voegelelein F., Mangatal L., Potier P. J., Am. Chem. Soc.,110:5917, 1988).

[0127] The reaction conditions for the preparation of Compound 3 weredetermined experimentally to be as follows. A solution of Compound 1(32.8 mg, 0.102 mmol) and di-2-pyridyl carbonate (DPC, 22.2 mg, 0.102mmol) in dry toluene (3.0 mL) was stirred at room temperature undernitrogen for 15 mins. Dimethylaminopyridine (DMAP, 4.2 mg, 0.034 mmol)and 7-triethyl silyl-10-deacetyl baccatin III (Compound 2) (12.0 mg,0.017 mmol) was added to the mixture. The reaction mixture was heated at72° C. for 16 hours, then allowed to cool to room temperature. Theorganic solvent was removed by rotary evaporation. The crude product wasdissolved in methanol (1.0 mL) and filtered. The solution of crudeproduct was purified by preparative reverse phase HPLC (50 cm) usinglinear gradient of acetonitrile (25% to 100%) in water over 70 minutesto give pure Compound 3 (R_(t)=77.6 min) as a white solid (10.5 mg,61.6% yield, R_(f)=0.51, EtOAc/Hexane =3/7). Analytical HPLC usinglinear gradient of acetonitrile (25% to 100%) in water over 50 minutesfor Compound 3 gave retention time (R_(t)=62.8 min). HRMS:MH⁺ requires:1004.48277, Found 1004.48280; ¹H-NMR of Compound 3: (500 MHz, CDCl₃) δ8.04 (d, J=7.3 Hz, 2H), 7.63 (t, J=7.3 Hz, 1H), 7.49 (t, J=7.8 Hz, 2H),7.40-7.30 (m, 5H, 3′-Ph), 6.44 (s, 1H, H-10), 6.23 (t, J=8.3 Hz, 1H,H-13), 5.65 (d, J=6.8 Hz, 1H, H-2), 5.04 (br., 1H, H-3′), 4.87 (d, J=8.8Hz, 1H, H-5), 4.46 (d, J=6.3 Hz, 1H, H-2′), 4.44 (o.m, 1H, H-7), 4.24(d, J=8.3 Hz, 1H, H-20a), 4.10 (d, J=8.3 Hz, 1H, H-20b), 3.77 (d, J=6.8Hz, 1H, H-3), 2.49 (ddd, J=14.6, 9.8, 6.3 Hz, 1H, H-6a), 2.20 (s, 3H,OAc), 2.15 (d, J=9.3 Hz, 2H,1H-14), 2.04 (s, 3H, Me-18), 1.87 (s, 3H,OAc), 1.82 (o.m, 1H, H-6b), 1.65 (s, 3H Me-19), 1.60 (br, t-Bu), 1.22(s, 3H, Me-16 or 17), 1.20 (s, 3H, Me-17 or 16), 1.11 (br, t-Bu), 0.57(s, 6H, Si—CH ₂), 0.91 (t, J=7.8 Hz, 9H, Si—CH₂₋ Me). This reaction wasrepeated another two times at the same scale. The yield of the later tworeactions were 51.2% and 68.0%. Thus, the yield of this reaction variesfrom 51.2%-68.0%.

[0128] Compound 3 (4.0 mg, 0.004 mmol) was stirred in 0.3 mL of 95-97%formic acid at room temperature for 4 hours. The formic acid was thenremoved by a flow of nitrogen and the crude product was dried undervacuum. The residue was dissolved in EtOAc (0.5 mL), and then NaHCO₃(10.0 mg) and isobutyryl chloride (5 μL, 0.047 mmol) were added. Afterstirring for 1 hour, EtOAc (10 mL) was added to the solution. Theorganic phase was washed with brine (3.0 mL) and dried over anhydrousMgSO₄. After filtration and evaporation, the residue was purified bypreparative HPLC (R_(t)=35.9 mins, acetonitrile from 25% to 100% inwater over 70 minutes). Compound 4 (Canadensol) was obtained as a whitesolid (46 μg, 1.4% yield based on Compound 3). Analytical HPLC usinglinear gradient of acetonitrile (25% to 100%) in water over 50 minutesfor Compound 4 gave retention time of 32.19 mins. HRMS: MH⁺ requires:820.35423, Found: 820.35443; ¹H-NMR of combined samples of Compound 4:(500 MHz, CDCl₃) δ 8.11 (d, J=7.8 Hz, 2H) 7.60 (t, J=7.3 Hz, 1H), 7.5(t, J=7.6 Hz, 2H), 7.40-7.30 (m, 5H, 3′-Ph), 6.27 (s, 1H, H-10), 6.20(t, J=9.5 Hz, 1H, H-13), 5.67 (d, J=6.5 Hz, 1H, H-2), 5.57 (d, J=8.8 Hz,1H, H-3′), 4.93 (d, J=9.1 Hz, 1H, H-5), 4.68 (o.m, 1H, H-2′), 4.39 (o.m,1H, H-7), 4.24 (d, J=8.8 Hz, 1H, H-20a), 4.18 (d, J=8.8 Hz, 1H, H-20b),3.78 (d, J=6.8 Hz, 1H, H-3), 3.40 (d, J=5.1 Hz, 1H, 2′—OH), 2.53 (ddd,J=15.4, 8.1, 7.2 Hz, 1H, H-6a), 2.38 (o.m, 1H Me₂ CHCONH), 2.35 (s, 3H,OAc), 2.28 (o.m, 2H, H-14), 2.24 (s, 3H, OAc), 1.84 (o.m, 1H, 6b), 1.81(s, 3H, Me-18), 1.67 (s, 3H, Me-19), 1.26 (s, 3H, Me-16 or 17), 1.15 (s,3H, Me-17 or 16), 1.11 (2d, J=6.5, 6.1 Hz, 6H Me ₂CHCONH). This reactionwas repeated two times. The yield of the later reaction was 1.8% and12.8%. Thus the yield of this reaction varies from 1.4%-12.4%.

EXAMPLE 2 Semi-Synthesis of Canadensol and a Structural Analogue

[0129] The preparation of Compound 1 was carried out through eight stepreactions from methyl cinnamate (as described in Denis J. D., Correa A.,Greene A. E., J. Org. Chem., 55:1959, 1990). The preparation of Compound5 was carried out through three step reactions from 10-deacetylbaccatinIII (as described in Denis J. N., Greene A. E., Guenard D.,Gueritte-Voegelelein F., Mangatal L., Potier P. J., Am. Chem. Soc.,110:5917, 1988). The reaction conditions for the preparation of Compound6 were determined experimentally to be as follows:

[0130] To a solution of Compound 5 (baccatin III) (78 mg, 0.133 mmol) inpyridine (1.5 mL) was added 2,2,2-trichloroethylchloroformate (62 μL;0.452 mmol) at 80° C. This mixture was stirred at 80° C. for 2.5 hours.Water (5 mL) was added and most of the pyridine was evaporated. Theaqueous phase was extracted with CH₂Cl₂ (3×20 mL). The organic phase wasdried over MgSO₄ and evaporated. The crude product was dissolved inmethanol (2.0 mL) and filtered. The solution of crude product waspurified by preparative HPLC using linear gradient of acetonitrile (25%to 100%) in water over 70 minutes to give pure Compound 6 (R₂ 52.0 min)as a white solid (36 mg, 25.5% yield, R_(f)=0.38, EtOAc/CH₂Cl₂=¼) andrecovery of Compound 5 (baccatin III). Analytical HPLC using lineargradient of acetonitrile (25% to 100%) in water over 50 minutes forCompound 6 gave a retention time of 44.0 mins. ¹H-NMR of Compound 6:(500 MHz, CDCl₃) δ 8.09 (d, J=7.6 Hz, 2H) 7.61 (t, J=7.2 Hz, 1H), 7.48(t, J=7.6 Hz, 2H), 6.38 (s, 1H, H-10), 5.62 (d, J=7.5 Hz, 1H, H-2), 5.58(m, 1H, H-7),5.02 and 4.63 (2d, J=12.5 Hz, 2H, CH ₂CCl₃),4.97 (d, J=9.3Hz, 1H, H-5),4.87 (m, 1H, H-13), 4.32 (d, J=8.6 Hz, 1H, H-20a), 4.14 (d,J=8.6 Hz, 1H, H-20b), 4.01 (d, J=6.8 Hz, 1H, H-3), 2.63 (ddd, J=15.0,8.5, 7.0 Hz, 1H, H-6a), 2.29 (s, 3H, OAc), 2.15 (d, J=9.3 Hz, 2H, H-14),2.04 (s, 3H, Me-18), 2.03 (m, 1H, H-6b) 1.82 (s, 3H, OAc), 1.58 (s, 3H,Me-19), 1.13 (s, 3H, Me-16 or 17), 1.09 (s, 3H, Me-17 or 16).

[0131] The reaction conditions for the preparation of Compound 7 weredetermined experimentally to be as follows: A solution of Compound 1(90.8 mg; 0.282 mmol) and di-2-pyridyl carbonate (DPC, 61.3 mg; 0.828mmol) in dry toluene (5.0 mL) was stirred at room temperature undernitrogen for 15 minutes. Dimethylaminopyridine (DMAP, 11.7 mg; 0.0954mmol) and 7-(2,2,2-trichloroethyloxycarbonyl)-baccatin III (Compound 6)(36.3 mg; 0.0476 mmol) was added to the mixture and stirred at 72° C.for 6 hours. The organic solvent was removed by rotary evaporation. Thecrude product was dissolved in methanol (1.5 mL) and filtered. Thesolution of crude product was purified by preparative HPLC using lineargradient of acetonitrile (25% to 100%) in water over 70 minutes to givepure Compound 7 (R_(t)=77.3 min) as a white solid (27.0 mg; 53.3% yield;R_(f)=0.43, EtOAc/hexane=1/1). Analytical HPLC using linear gradient ofacetonitrile (25% to 100%) in water over 50 minutes for Compound 7 gaveretention time (R_(t)=56.2 min). ¹H-NMR of Compound 7: (500 MHz, CDCl₃)δ 8.04 (d, J=7.3 Hz, 2H), 7.64 (t, J=7.3 Hz, 1H), 7.50 (t, J=7.8 Hz,2H), 7.40-7.30 (m, 5H, 3′-Ph), 6.36 (s, 1H H-10), 6.26 (br.t, J=8.5 Hz,1H, H-13), 5.65 (d, J=7.1 Hz, 1H, H-2), 5.58 (dd, J=10.7, 7.1 Hz, 1H,H-7), 5.04 (br., 1H, H-3′), 5.03 and 4.64 (2d, J=12.0 Hz, 2H CH ₂CCl₃),4.91 (dd, J=9.7, 1.5 Hz, 1H, H-5), 4.47 (d, J=6.9 Hz, 1H, H-2′), 4.27(d, J=8.5 Hz, 1H, H-20a), 4.11 (d, J=8.5 Hz, 1H, H-20b), 3.93 (d, J=7.1Hz, 1H, H-3), 2.59, (ddd, J=14.6, 9.7, 7.1 Hz, 1H, H-6a), 2.18 (om, 2H,H-14), 2.17 (s, 6H, 2OAc), 2.03 (o.m, 1H, H-6b), 2.00 (d, J=1.0 Hz, 3H,Me-18), 1.91 and 1.80 (2s, 6H, Me ₂CNO), 1.76 (s, 3H Me-19), 1.24 (s,3H, Me-16 or 17), 1.16 (s, 3H, Me-17 or 16), 1.10 (br, t-Bu).

[0132] The reaction conditions for the preparation of Compound 8 weredetermined experimentally to be as follows: Compound 7 (26.0 mg; 0.0244mmol) was stirred in formic acid (2.0 mL) at room temperature for 4hours. Formic acid was then removed by a flow of nitrogen and crudeproduct was obtained under vacuum. The residue was redissolved in EtOAc(3.0 mL). Then, NaHCO₃ (61 mg; 0.726 mmol) and isobutyryl chloride (26μL; 0.0366 mmol) were added. After being stirred for 1 hour EtOAc (20mL) was added to the mixture. The organic phase was washed with asaturated solution of NaCl (5 mL) and dried over MgSO₄. After filtrationand evaporation, the residue was purified by preparative HPLC usinglinear gradient of acetonitrile (25% to 100%) in water over 70 minutesto give pure Compound 8 (R_(t)=55.8 min) as a white solid (12.0 mg;50.0% yield; R_(f)=1.19; EtOAc/Hexane=1/1). Analytical HPLC using lineargradient of acetonitrile (25% to 100%) in water over 50 mins forCompound 8 gave retention time (R_(f)=46.1 min). ¹H-NMR of Compound 8:(500 MHz, CDCl₃) δ 8.10 (d, J=7.8 Hz, 2H), 7.62 (t, J=7.3 Hz, 1H), 7.51(t, J=7.6 Hz, 2H), 7.43-7.34 (m, 5H, 3′-Ph), 6.35 (s, 1H H-10), 6.26 (d,J=9.1 Hz, 1H, NH-4′), 6.16 (br.td, J=9.0, 1.3 Hz, 1H, H-13), 5.69 (d,J=7.1 Hz, 1H, H-5), 5.58 (dd, J=9.0, 2.4 Hz, 1H, H-3′), 5.54 (dd,J=10.5, 7.3 Hz, 1H, H-7), 5.03 and 4.64 (2d, J=12.0, 12.0 Hz, 2H, CH₂CCl₃), 4.95 (br.d, J=9.5 Hz, 1H, H-5), 4.70 (dd, J=4.8, 2.4 Hz, 1H,H-2′), 4.31, (d, J=8.6 Hz, 1H, H-20a), 4.18 (d, J=8.5 Hz, 1H, H-20b),3.92 (d, J=7.0 Hz, 1H, H-3), 3.42 (d, J=4.9 Hz, 1H, OH-2′), 2.61 (ddd,J=14.4, 9.8, 7.1 Hz, 1H, H-6a), 2.40 (o.septet, J=6.8 Hz, 1H, Me₂CHCONH), 2.36 (s, 3H, OAc), 2.33 (dd, J=9.0, 1.4 Hz, 2H, H-14),2.17 (s,3H, OAc), 2.05 (m, 1H, H-6b), 1.88 (s, 3H, Me-18), 1.84 (s, 3H, Me-19),1.24 (s, 3H Me-16 or 17), 1.18 (s, 3H, Me-17 or 16), 1.13 and 1.12 (2d,J=6.8, 6.8 Hz, 6H, Me ₂CHCONH).

[0133] The reaction conditions for the preparation of Compound 4 weredetermined experimentally to be as follows: To a solution of Compound 8(11.7 mg; 0.0117 mmol) in acetic acid:methanol (1:1, v/v, 2 mL) wasadded zinc powder (70.6 mg) at 60° C. under nitrogen. This mixture wasstirred at 60° C. under nitrogen for 1 hour. Then CH₂Cl₂ (20 mL) wasadded and the mixture was filtered through celite. The organic solventwas evaporated to give crude product. The residue was purified bypreparative HPLC using linear gradient of acetonitrile (25% to 100%) inwater over 70 minutes to give pure Compound 4 (R_(t)=37.0 min) as awhite solid (7.2 mg; 74.7% yield). Analytical HPLC using linear gradientof acetonitrile (25% to 100%) in water over 50 minutes for Compound 4gave retention time (R_(t)=33.0 min). HRMS: MH⁺ requires: 820.35423,Found: 820.35443; ¹H-NMR of Compound 4: (500 MHz, CDCl₃) δ 8.12 (d,J=6.8 Hz, 2H) 7.61 (t, J=7.3 Hz, 1H), 7.51 (t, J=7.6 Hz, 2H), 7.44-7.30(m, 5H, 3′-Ph), 6.28 (s, 1H, H-10), 6.24 (d, J=9.0 Hz, 1 h NH-4′), 6.20(t, J=8.5 Hz, 1H, H-13), 5.68 (d, J=7.1 Hz, 1H, H-2), 5.57 (d, J=9.0,2.5 Hz, 1H, H-3′), 4.94 (dd, J=9.8, 2.2 Hz, 1H, H-5), 4.69 (d, J=2.7 Hz,1H, H-2′), 4.40 (dd, J=11.2, 5.8 Hz, 1H, H-7),4.29 (d, J=8.5 Hz, 1H,H-20a), 4.19 (d, J=8.5 Hz, 1H, H-20b), 3.78 (d, J=7.1 Hz, 1H, H-3), 2.54(ddd, J=14.9, 9.8, 6.9 Hz, 1H, H-6a), 2.39 (o.septet, J=6.8 Hz, 1H, Me₂CHCONH), 2.35 (s, 3H, OAc), 2.28-2.32 (m, 2H, H-14), 2.24 (s, 3H, OAc),1.88 (ddd, J=13.4, 11.0, 2.2 Hz, 1H, H-6b), 1.82 (s, 3H, Me-18), 1.68(s, 3H, Me-19), 1.26 (s, 3H, Me-16 or 17), 1.15 (s, 3H, Me-17 or 16),1.12 and 1.11 (2d, J=7.0, 6.8 Hz, 6H Me ₂CHCONH).

EXAMPLE 3 Semi-Synthesis of N-debenzoyl-N-n-pentanoyl-paclitaxel

[0134] Following the same method of synthesis as described in Example 2,alternatively, Compound 7 can be transformed into a family of relatedtaxanes by the use of different alkyl chlorides. Specifically, Compound7 can be transformed into a family of related taxanes by the use ofdifferent acyl chlorides, for example:

[0135] Compound 7 (12 mg; 0.011 mmol) was stirred in formic acid (0.5mL) at room temperature for 4 hours and the solution was then evaporatedwith a stream of nitrogen. Ethyl acetate (1.5 mL), sodium bicarbonate(15 mg; 0.18 mmol) and propanoyl chloride (14.5 μL; 0.12 mmol) wereadded and the mixture stirred for 1 hour at room temperature. Ethylacetate (20 mL) was added and the solution was washed with saturatedsodium chloride, dried over anhydrous magnesium sulphate and evaporated.The residue was purified on preparative HPLC using a linear gradient ofacetonitrile (25% to 100%) in water over 70 minutes to yield Compound 9(Rt=57.5min) as a white solid (8.5 mg; 77% yield). Analytical HPLC usinga linear gradient of acetonitrile (25% to 100%) in water over 50 min forCompound 9 gave a retention time of 47.3 min.

[0136] Compound 9 (7.0 mg; 0.0072 mmol) was dissolved in 3.0 mL of asolution of methanol:acetic acid (1:1; v/v) and zinc (30.0 mg; 0.46mmol) was added. The mixture was stirred at 60° C. for 1 hour. Themixture was then left to cool at room temperature and thendichloromethane (20 mL) was added. Filtration over celite andpurification on preparative HPLC using a linear gradient of acetonitrile(25% to 100%) in water over 70 minutes (Rt=42.5 min) afforded Compound10 as a white solid (2.6 mg; 44% yield).

[0137] Analytical HPLC using a linear gradient of acetonitrile (25% to100%) in water over 50 minutes for Compound 10 gave a retention time of34.8 min. HRMS with KI as an internal standard: expected molecularweight; 872.32596 obtained 872.32574.

EXAMPLE 4 Semi-Synthesis of Canadensol (Scheme III)

[0138] The semi-synthesis of canadensol, taxcultine or any of the otherbiological active compounds can be obtained using baccatin III asstarting material. The conversion of the natural product10-deacetylbaccatin III into baccatin III can be achieved according tothe technique described in Denis J. N. et al., J. Am. Chem. Soc., 110:5917 (1988). The conversion of baccatin III into canadensol requiresthat the hydroxyl at C-7 be protected prior to derivatization with theappropriate side chain at C-13. An appropriate side chain is thetriethylsilyl derivative of the C-7 hydroxyl). For the supply of largeamounts of canadensol, taxcultine, etc, the addition of the side chainsat C-13 is achieved in higher yields with the use of a range of sidechains by Ojima's method (Ojima, I. et al., Tetrahedron, 48. 6985-7012,1992; and Ojima, I. et al., Tetrahedron Letters, 34, 4149-4152, 1992).

[0139] The β-lactam (1) was obtained from (3R,4S)-3-hydroxy-4-phenylazetidin-2-one (which was prepared according to Ojima,I. et al Tetrahedron, 48, 6985-7012, 1992) by treatment with tetrabutylammonium fluoride according to the following conditions:

[0140] (3R,4S)-3-triisopropylsilyloxy-4-phenylazetidin-2-one (0.200 g;0.626 mmol) was dissolved in dry tetrahydrofuran (4 mL) under nitrogenat room temperature. To this was added a solution of a n-Bu₄N⁺F⁻ (1.0Min dry tetrahydrofuran; 1.2 ml; 1.2 mmol; 1.9 eq.) and stirred for 2.5hours at room temperature. The reaction was complete as shown by thinlayer chromatography with 35% ethylacetate/hexane which separated verywell the starting material from the product. The work up consisted of anextraction with ethyl acetate, washing with brine, drying the ethylacetate layer with magnesium sulfate then evaporated. The product (0.12g) was obtained with almost 100% yield after flash chromatography.

[0141] The next step in the synthesis is protection of the hydroxy groupof the β-lactam with triethylsily chloride according to the followingconditions:

[0142] The β-lactam (1) (0.12 g; 0.735 mmol) was dissolved in 10 mlpyridine at room temperature under nitrogen. The triethylsilyl chloride(0.40 ml; 2.38 mmol; 3.2 eq) was added at room temperature and thereaction was complete after one hour at room temperature. The work upconsisted on quenching the reaction with saturated ammonium chloride,extract with methylene chloride, wash the methylene chloride solutionwith brine and dry the methylene chloride layer with magnesium sulfate.After filtration the solvent was evaporated. Most of the pyridine whichwas left behind was evaporated with heptane before running a flashchromatography with a 7.5″ small column with 30% ethylacetate/hexane andcollected the fractions 24-45(0.129 g). The yield of the combined twosteps is 74%. The NMR was in accord with structure 2.

[0143] Next step is specific to the side chain we want to add. Here wewill show the experimental for the side chain of canadensol and in thenext page the coupling reaction with the protected baccatin derivative.

[0144] The triethyl-silyl-protected β-lactam (2) (0.030 g: 0.108 mmol)was dissolved in 1.5 ml dry methylene chloride under nitrogen at roomtemperature, triethylamine (41 μl; 0.294 mmol; 2.7 eq.) a trace of4-DMAP (4-dimmethylaminopyridine), the solution was cooled in an icewater bath and isobutyryl chloride (0.025 ml; 0.239 mmol; 2.2 eq.) wasstirred at room temperature for 3 hours when the reaction was complete.The work up consisted on quenching the reaction with saturated ammoniumchloride, extract with methylene chloride, wash the methylene chloridesolution with brine and dry the methylene chloride layer with magnesiumsulfate. After filtration the solvent was evaporated and flashchromatography with 25% ethyl acetate/hexane in a 6″ chromatographycolumn 0.034 g of the required β-lactam (90% yield) (3) for the semisynthesis of canadensol was obtained.

[0145] Coupling reaction: the C-7 triethylsilylprotected baccatin III(0.048 g: 0.0685 mmol) and the triethylsily-isopropyl-β-lactam (3)(0.043 g; 0.0978 mmol; 1.4 eq.) were dissolved in 3.6 ml drytetrahydrofuran. The solution was cooled to a temperature in the range−45° C. to −50° C. (dry ice-acetonitrile bath) under nitrogen. The basesodium bistrimethylsilylamide (NaHMDS) (1.0 M) solution intetrahydrofuran; 0.175 ml; 175 mmol; 2.6 eq) was added in one portion ata temperature of −45° C. and the reaction was stirred for 30 min at thistemperature. The solution became yellow and was quenched at 30 min withan ammonium chloride saturated solution. The work up consisted ofquenching the reaction with saturated ammonium chloride, extracting withethyl acetate, washing the organic phase with brine and drying theorganic layer with magnesium sulphate. After filtration the solvent wasevaporated and flash chromatography of the residue with 20% ethylacetate/hexane in a 6″ chromatography column; 36 mg of the coupledprotected taxane (5) was obtained with 14 mg of starting materialrecovered (4, uncoupled protected baccatin III).

[0146] Preparation of Canadensol

[0147] The deprotection of compound 5 was completed with 0.3 M HCl in95% ethanol at 4° C. overnight. At this stage the reaction was complete,the reaction mixture cooled in an ice-water bath; ice added to reactionmixture and the pH adjusted to 5-6 with the dropwise addition of asaturated sodium bicarbonate solution. The reaction mixture is extractedfour times with ethyl acetate, the organic phase washed with brine, theorganic layer dried with MgSO₄, filtered and the solvent evaporated. Theresidue was subjected to flash chromatography with 75% ethylacetate/hexane in a 7″ mini column and 0.024 g of canadensol (6) wasobtained with identical NMR and high resolution mass spectrometry as thecanadensol obtained by Denis et al. method.

EXAMPLE 5 In Vitro Microtubule Assay

[0148] Tubulin is obtained by the purification of calf brain (WilliamsJr, R. C. and J. C. Lee, Methods in Enzymology, 85:376, 1982). Thetaxanes to be tested are dissolved in dimethylsulfoxide (DMSO) with afinal concentration of 10 μM. The methodologies used in this microtubuleassay are well known to those skilled in the art. The method brieflyconsists of mixing the buffer and other required ingredients in acuvette in an ice bath, with the DMSO solution containing the taxane tobe analysed. Before the start of the measurement, the tubulin is addedand the cuvette is set in a thermostatically controlled cell compartmentof a UV-VIS spectrophotometer. The temperature is set at 37° C. to startthe polymerisation of the tubulin. The control is performed with all thesame ingredients just without the added taxane. Since bio-active taxanescause tubulin to polymerize to microtubules and stabilize them, theturbidity of the mixture will rise and causing a corresponding increasein the absorbance observed at 350 nm. Theoretically, the faster theincrease in absorbance, the more active the taxane.

[0149] As shown in FIGS. 1-4, Canadensol is more active than paclitaxeland taxcultine in these tests. FIG. 1 illustrates that, at the sameconcentration, semi-synthetic Canadensol (ssC) causes higher degree oftubulin polymerisation than Paclitaxel (P).

[0150]FIG. 2 illustrates that 5 μM semi-synthetic Canadensol (ssC)appears to cause a similar degree of tubulin polymerisation as 10 μMPaclitaxel (P), this figure also emphasizes the dose-dependent responseof tubulin polymerisation to semi-synthetic Canadensol (ssC) atconcentrations of 5 μM and 10 μM.

[0151]FIG. 3 illustrates that, at the same concentration (10 μM),semi-synthetic Canadensol (ssC) exhibits greater enhancement of tubulinpolymerisation than semi-synthetic Taxcultine (ssT) or paclitaxel (P).

[0152]FIG. 4 illustrates that semi-synthetic Canadensol (ssC) obtainedby the method described in the preferred embodiment of this inventionshows similar activity to a mixture of 13% Canadensol and 87% Taxcultinepurified from Taxus canadensis.

EXAMPLE 6 Preclinical Studies with Taxoids

[0153] Methods

[0154] 1—Cell lines and cell culture. The human ovarian adenocarcinomacell line A2780 was used to evaluate the antiproliferative activity oftaxoids. These cells were grown in either RPMI medium supplemented with10% fetal bovine serum and penicillin-streptomycin antibiotics. Cellswere maintained in culture at 37° C. in an atmosphere of 5% CO₂.

[0155] 2—Cytotoxicity assay. Exponentially growing cells (2-3×10³cells/100 ml) were seeded in 96-well plates and incubated for 16 h.Cells were then treated continuously with the extracts. 72 h later, cellsurvival was evaluated by replacing the culture media with 150 ml freshmedium containing 10 mM 4-(2-hydroxyethyl)-1 -piperazineethamesulfonicacid buffer, pH 7.4. and 50 ml of 2.5 mg/ml of3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) inPBS, pH 7.4, were then added. After as 3-4 h of incubation at 37° C.,the medium and MTT were removed, and 200 ml of DMSO was added todissolve the precipitate of reduced MTT, followed by addition of 25 mlglycine buffer (0.1M glycine plus 0.1M NaCl, pH 10.5). The formazancrystals were then dissolved and the absorbance was determined at 570 nmwith a microplate reader (BIORAD, model 450). The MTT assaydistinguished between viable and non-viable cells on the basis for therequirement of physiologically active mitochondria to metabolize the MTTonly in viable cells. The IC₅₀ was calculated as the concentration ofdrug causing a 50% inhibition in the absorbance compared to cellstreated with solvent alone, Table I.

[0156] In Vivo Study Using GA3 Cell Model

[0157] Origin: The DA3 cell line used in this study was derived from ahyperplastic mammary outgrowth (preneoplastic lesion) treated with7,12-dimethylbenzanthracene (DMBA). DMBA-treated explant was thentransplanted into the mammary glands of a female BALB/c mouse, and theresulting tumor was used to establish the DA3 cell line. DA3 is animmunogenic and non-metastatic mammary adenocarcinoma.

[0158] Source: The cell line used in our study was originally obtainedfrom Dr. Medina in 1993. Stocks of these cells were generated and storedduring early passages in liquid nitrogen. One vial from this early stockwas cultured and cells generated were used to confirm the absence ofviral and mycoplasma infections. Cells were then propagated and stocksof these cells were established and stored in liquid nitrogen forfurther studies with Dup compounds.

[0159] Cell culture: DA3 cells were maintained in RPMI 1640 mediumsupplemented with 1M mercaptoethanol, 1M Hepes buffer solution, 100 nMsodium pyruvate, 200 mM L-glutamine, 10 mM non-essential amino acids, 1Mvitamins, 10% fetal bovine serum, 1% penicillin-streptomycin. Cells weremaintained at 37° C. under 5% CO₂. Under these conditions, DA3 cellsproliferate but do not differentiate.

[0160] Animal. Female BALB/c mice (Charles River Inc.) were used to growDA3 tumors. Mice were maintained at the Lady Davis Institute [LDI]Animal Care Facility. The animal facility is located in the basement ofthe LDI and is completely isolated from the research laboratories andadministrative offices. Animal rooms are segregated from surgery rooms,offices, autoclaves, incinerators, and access to the animal facility isstrictly limited to animal users only. Animal room used in our studieshas two doors, one serving as the entrance, and the other door providesdirect access to washing/sterilization/incineration facilities. Itpermits accurate adjustment of environmental parameters includingtemperature, humidity, ventilation, and lighting. Cleaning andsanitation practices are performed, on a daily basis, by personnel withappropriate training.

[0161] Tumor cell inoculation and treatments. After one weekacclimatisation, mice were randomized into a group of 5 per cage. Cageswere randomly assigned to specific experimental groups. The mice werethen labeled by numbers using the “ear punching” method. DA3 cells weretransplanted subcutaneously to mice, as a suspension of tumor cells[1×10⁶ viable cells per 0.1 ml], in the right flank. All animals wereinoculated at the same site.

[0162] For tumor induction, cells were grown to 70% confluence incomplete medium and then collected using trypsin-EDTA solution [0.05%trypsin, 0.53 mM EDTA-4Na in HBSS Ca⁺⁺, Mg⁺⁺ and NaHCO₃ free]. cellswere then centrifuged and washed three times with phosphate buffersolution and resuspended at a dilution of 0.1 to 1×10⁶ cells/0.1 ml.Viability was examined by trypan blue staining and only flasks in whichthe viability was ≧95% were used for in vivo studies.

[0163] Treatment was initiated when tumors become palpable. Drug wasgiven twice (day 1 and day 3) by iv. Control animals were given the samevolume of saline solution. The dose of each drug was normalized to mousebody weight.

[0164] Tumor measurement. animals were examined every day but the tumorgrowth was monitored every second or third day using calipers.Parameters measured are: tumor measured along the longest axis (length)and the perpendicular shortest axis (width) and the relative tumorvolume (in cm³) was calculated by the formula: [Length (cm)×(widthcm)²]/2. Tumors which reach the limit size permitted by the Canadianregulation on the use of laboratory animals, or showing any apparentdistress or discomfort were immediately sacrificed independently of theexperimental protocol [usually 2-3 cm³ but this depend on animal statusand the decision is taken by the animal room personnel]. Animals weresubjected, on a daily basis, to general examination.

[0165] Statistical analysis. The unpaired Student t-test was used tocompare statistical significance among various groups.

[0166] Results

[0167] In Vitro Studies

[0168] Canadensol was found to be as active as paclitaxel or taxcultine,in inhibiting the growth of the human ovarian carcinoma cell line,A2780, as shown in FIGS. 6, 7 and Table I.

[0169] In Vivo Studies

[0170] The mouse mammary adenocarcinoma tumors were resistant topaclitaxel since no significant antitumor activity was observed withtolerable doses (20-40 mg/Kg). Higher doses of paclitaxel were found tobe very toxic (body weight loss and mortality), see FIG. 8B. Canadensol,however, was found to induce antitumor activity when given twice at 40mg/kg, see FIG. 9. This effect was significant from day 30 to day 40 oftumor growth. No toxic effect was observed at this dose. TABLE I A2780Conc. (nM) Abs. % IC₅₀ TAXOL  0 1.433 100.00%  0.2 1.455 101.54%  21.464 102.16%  10 1.162 81.09% 16.96  20 0.522 36.43% 100 0.021 1.47%200 0.02 1.40% CANADENSOL  0 1.308  0.2 1.598 100.00%  2 1.504 94.12% 10 1.246 77.97%  20 0.812 50.81% 21.32 100 0.026 1.63% 200 0.018 1.13%TAXCULTINE  0 1.155  0.2 1.535 100.00%  2 1.536 100.07%  10 1.093 71.21%16.04  20 0.554 36.09% 100 0.032 2.08% 200 0.022 1.43%

We claim:
 1. A compound of formula III:

wherein R₃ is selected from the group consisting of propyl, isopropyland n-butyl.
 2. The compound as in claim 1, wherein R₃ is isopropyl. 3.A compound of formula IV:

wherein R₃ is selected from the group consisting of propyl, isopropyland n-butyl.
 4. The compound as in claim 3, wherein R₃ is isopropyl. 5.A compound of formula V:

wherein R₄ is SiEt₃ or CO₂CH₂CCl₃.
 6. A process for the preparation of acompund of formula V, which comprises the steps of: (i) protecting ahydroxy group at the 7 position of baccatin III:

to form a 7-OH-protected baccatin III; and (ii) reacting the7-OH-protected baccatin III with a compound of formula VII.


7. The process of claim 6, wherein the hydroxy group at the 7 positionis protected with SiEt₃ or CO₂CH₂CCl₃.
 8. A method of inhibiting tumoursin a mammal in need of such treatment, comprising administering to themammal a therapeutically effective amount of a compound of formula III:

wherein R₃ is selected from the group consisting of propyl, isopropyland n-butyl.
 9. The method of claim 8, wherein R₃ is isopropyl.
 10. Apharmaceutical formulation comprising one or more compounds of formulaIII:

and a pharmaceutically acceptable carrier; wherein R₃ is selected fromthe group consisting of propyl, isopropyl and n-butyl.
 11. Thepharmaceutical formulation of claim 10, wherein R₃ is isopropyl.